Papillomaviruses (PVs) infect the epithelia of animals and man, where they generally induce benign proliferation at the site of infection. However, there is a strong association between malignant progression of human genital, anal and oropharyngeal lesions and certain human papillomavirus (HPV) types, most frequently HPV 16. Our research is primarily concerned with development of vaccines and other infection inhibition strategies against HPV and the elucidation of the HPV life cycle. We have developed a simple and efficient strategy for generating high titers of infectious papillomavirus particles that transduce encapsidated marker plasmids, i.e. pseudovirions. We have exploited this technology in our basic virologic and translational research efforts. We have used our pseudovirus technology to develop the first cervicovaginal challenge model for HPVs. We found that the infection of the female mouse genital tract, even of monolayer endocervical cells, requires exposure the basement membrane to the virus. The capsids bind avidly to the basement membrane but not to the apical surfaces of intact columnar or stratified squamous epithelia. In a process unique to papillomaviruses, we showed that the initial steps in infection occur after initial binding to heperan sulfate proteoglycans (HSPG) on the basement membrane, prior cell surface binding. Specifically the virions undergo a conformational change that exposes the N-terminus of the L2 minor capsid protein to cleavage by furin, a cellular protease that we detect in abundance at the sites of infection in vivo. This cleavage in turns leads to exposure of highly conserved L2 cross-neutralizing epitopes that are immediately downstream of the cleavage site. Blocking HSPG interactions or furin cleavage prevented cerviovaginal infection. In a process that takes several hours, the virions transfer from the basement membrane to the surface of keratinocytes invading the site of trauma, and the virions are then internalized. These finding led to our determining the mechanisms of in vivo protection conferred by VLP and L2 specific antibodies and, as described, below, led to our developing more sensitive assays for quantifying protective antibodies. In a collaborative study with Denise Nardelli's group, we showed that, surprisingly, our cervicovaginal challenge assay is more than a 100-fold more sensitive measure of VLP induced protective antibodies than is our standard in vitro neutralizing assay. The results imply that detection of even low levels of in vitro neutralizing antibodies in Gardasil and Cervarix vaccinees may predict strong and durable protection. The in vitro neutralizing assays we previously developed for L1 VLP vaccine analysis have proven to be relatively insensitive measures of protective L2 antibodies. Based on our understanding of the in vivo infectious process, we have recently developed a novel in vitro neutralizing assay that is 1000-fold more sensitive measure of L2 antibody activity. This assay will be critical in the further clinical development of L2-based vaccines, which we previously discovered to induce antibodies that, unlike L1 VLP vaccines, broadly cross-neutralize divergent mucosal and cutaneous HPV tpes.Our development of a method to induce efficient HPV pseudovirus infection of the female genital tract after transient disruption with the over-the-counter spermicide nonoxonol-9 has proven to be the key to our recent development of an effective, and we believe practical, intravaginal vaccination strategy. We have found that intravaginal pseudovirus vaccination of N-9 treated mice induces strong systemic and mucosal T and B cell responses to target antigens transduced by the pseudovirions. Systemic responses rival those induced by previously optimized Ad5 vectors. Intravaginal responses are remarkably strong, with up to 80% of all intravaginal CD8 T cells staining tetramer positive for the targeted antigen. Most of the induced T appear to be intraepithelial, and high level of effector memory CD8 T cells are maintained in the vaginal tract 100 days after vaccination. Critically, CD8 IEL's were not induced after systemic vaccination with Ad5 vectors. A manuscript detailing these studies is under final revision for publication in the Journal of Clinical Investigation. Intravaginal pseudovirus vaccination is a promising approach for focusing immune responses to the female genital tract and so might increase the effectiveness of vaccines directed against HSV and HIV infections and against HPV induced neoplasia. This concept has been being tested in an SIV/rhesus macaque intravaginal challenge model in collaboration with Dr. Franchini. In collaboration with Dr. Jeff Cohen, vectors expressing HSV antigens are being tested in mouse and guinea pig HSV-2 challenge models.To more generally evaluate the potential of HPV pseudoviruses as gene transfer vehicles, we have conducted a broad infection tropism survey. In patent pending studies, we demonstrated that intact murine epithelium at all sites, whether simple, columnar, or squamous, was highly resistant to both virion binding and infection, whereas disrupted epithelium was susceptible. In contrast, virtually all human-derived epithelial cell lines in the NC1-60 panel were highly susceptible to infection in vitro. The remarkable specificity of HPV pseudovirus binding and infection appears to be mediated by specific heparan sulfate modifications of proteoglycan on the tumor cell surfaces that mimic those normally found on the basement membrane. The resuts suggest that HPV pseudovirions may be useful in tumor diagnostic or cytotoxic gene therapy applications. In proof of concept studies, we documented highly specific binding and infection, and dramatic imaging, of human ovarian tumor nodules implanted in nude mouse peritoneum after intraperitoneal injection of RFP-expressing pseudovirus. In a preliminarly study using a mouse model of ovarian metastases, intraperitoneal injection of Herpes TK-expressing HPV psuedovirions followed by ganciclovir treatment, increased survival of tumor bearing mice. A manscript reporting these findings is being prepared. A CRADA with Aura Biosciences was recently signed to facilitate further development and clinical testing of this approach to tumor therapy. A long standing collaboration with DCEG colleagues have resulted in several publications related to the NCI-sponsored prophylactic HPV vaccine clinical trial in Costa Rica. Highlights include demonstration of protection from persistent cervical infection by vaccine-targeted and related types, strong vaccine-type specific protection for four years in women receiving only one or two doses of the vaccine, and protection from anal infection by the vaccine targeted types.